There are known a variety of techniques for the preparation of nucleotide oligomers.
For example, methods of preparing the nucleotide oligomers can be found in the following references: Khorana et al., J. Molec. Biol. 72:209 (1972); Reese, Tetrahedron Lett. 34:3143 (1978); Beaucage and Caruthers, Tetrahedron Lett. 22:1859 (1981); U.S. Pat. No. 5,149,798; Agrawal and Goodchild, Tetrahedron Lett. 28:3539 (1987); Connolly et al. Biochemistry 23, 3443 (1984); Jager et al., Biochemistry 27:7237 (1988); Agrawal et al. Proc. Natl. Acad. Sci. USA 85:7079 (1988), e.g., Methods in Molecular Biology, Vol. 20, Protocols for Oligonucleotides and Analogs, p. 63-80 (S. Agrawal, Ed., Humana Press 1993); Methods in Molecular Biology, Vol. 26: Protocols for Oligonucleotide Conjugates (Agrawal, Ed., Humana Press, Totowa, N.J. 1994); Oligonucleotides and Analogues: A Practical Approach pp. 155-183 (Eckstein, Ed., IRL Press, Oxford 1991); Antisense Res. and Applns. pp. 375 (Crooke and Lebleu, Eds., CRC Press, Boca Raton, Fla. 1993); and Gene Regulation: Biology of Antisense RNA and DNA (Erickson and Izant, eds., Raven Press, New York, 1992).
Anti-sense RNA hybridizes to nucleic acid molecules to result in the inhibition of gene expression. Many researchers have reported the inhibition of expression of specific genes or therapeutic feasibility of particular diseases via the use of the antisense RNA (Barker et al. Proc. Natl. Acad. Sci. USA 93:514 (1996); Agrawal et al., Proc. Natl. Acad. Sci. USA 85:7079 (1988); Letter et al., Proc. Natl. Acad. Sci. USA 87:3420-3434 (1990); and Offensperger et al. EMBO J. 12:1257 (1993)).
Meanwhile, RNA-mediated interference (RNAi) is a phenomenon in which a 21-25-nucleotide small RNA fragment selectively binds to and degrades mRNA having a complementary sequence, thus resulting in the suppression of protein expression (Shen C, et al., FEBS Lett. 539 (1-3):111-4 (2003)). The RNAi phenomenon was first discovered in 1995 as a part of the gene-regulation mechanism in Caenorphabditis elegans and plants. In 1998, Dr. Andrew Fire of the Carnegie Institution of Washington and Dr. Craig Mello of the University of Massachusetts Medical School, and their team experimentally found that the expression of a specific gene can be significantly inhibited when double-stranded RNA (dsRNA) corresponding to a base sequence of the specific gene is in-vivo injected into C. elegans (Fire A, et al., Nature. 391 (6669):806-11 (1998)). The long-chain dsRNA injected into C. elegans is cleaved into a short double-stranded RNA fragment called small interfering RNA (siRNA) about 21-25 by long, by the enzymatic action of Dicer belonging to a member of the RNase III family of nucleases which specifically cleave double-stranded RNAs. The resulting short dsRNA is then incorporated into the RNA-induced silencing complex (RISC) where the siRNA duplex is unwound into two strands. Thereafter, the siRNA separated into single-strands binds to a specific gene mRNA with a complementary sequence and makes it untranslatable, thus inhibiting the expression of the corresponding gene. Further, Elbashir and his colleagues have reported that the expression of a specific gene can be selectively inhibited by injection of short dsRNA (siRNA) consisting of 21 bases into cultured mammalian cells, this finding leading to significant increases in practical applicability of RNAi in mammalian cells (Elbashir, S. M. et al., Nature 411 (6836):494-8 (2001)).
At present, siRNA-mediated gene expression inhibition techniques are widely used in functional understanding of various genes and a great deal of research has been actively focused on exploitation of such siRNAs for development of therapeutic agents for the treatment of intractable diseases such as cancers, infectious diseases, etc. (Mouldy Sioud. Therapeutic siRNAs. Trends in pharmacological Sciences 2004; 22-28).
As discussed above, many attempts have been made to develop therapeutic agents or diagnostic agents using antisense RNAs and siRNAs. To this end, there is an urgent need for an efficient mass production scheme of oligoribonucleotides.
Synthesis of nucleotide oligomers is usually carried out by sequential coupling of monomer units on solid resins, using an automatic DNA/RNA (or oligonucleotide) synthesizer. DNA oligomers can be synthesized with a good yield. On the other hand, synthesis of RNA oligomers, e.g. ribonucleotide oligomers entails various disadvantages due to steric hindrance of a protecting group for a 2′-OH group, such as long synthesis period and low coupling efficiency resulting in low production yield, thus making it difficult to obtain high-purity RNA oligos.
Throughout the specification, numerous scientific articles and patent publications are cited and citations thereof are identified. Disclosures of the cited articles and patent references are incorporated by reference herein in their entirety, such that a current status of a technical field to which the present invention pertains and the disclosure of the present invention will be more clearly described.